Homogeneous multiplex screening assays and kits

ABSTRACT

Highly multiplexed homogeneous in vitro screening assays for numerous possible nucleic acid targets, any of which might be present in a sample, utilize fluorescent hybridization probes that are combinatorially coded from a panel of fluorophores by subdividing each probe into portions and differently labeling each portion such that, when portions are combined, each probe has a unique code. The assays may include target amplification and real-time detection. Probe sets and kits containing additional assay reagents may be used to perform the screening assays.

TECHNICAL FIELD

This invention relates to homogenous in vitro multiplexed assays,including particularly homogeneous amplification assays, for screeningbiological samples for the presence of any of a large number of nucleicacid sequences.

BACKGROUND

A variety of assay techniques are available for testing a biologicalsample obtained from any of a variety of sources for the presence of anucleic acid sequence that may indicate the presence, for example, of aparticular bacterium, virus or other pathogen, including a particularstrain or mutant. Assays are also available for testing such samples forthe presence of a nucleic acid sequence of the subject's own genomic DNAthat may indicate the presence, for example, of one or anotherdisease-related genetic mutation. Assays may include oligonucleotideprobes bearing detectable labels, for example, P³² or fluorophores.Nucleic acids, either DNA or RNA, in a sample may be probed directly.Alternatively, assays may include amplification of target sequences byany of several amplification techniques, for example, PCR, NASBA or TMA.Amplification assays may be monitored in real time utilizingintercalating dyes, for example SYBR green, or fluorescently labeledprobes, such as 5′ nuclease probes Livak, K. J. et al. (1995),Oligonucleotides with fluorescent Dyes at Opposite Ends Provide aQuenched Probe System Useful for Detecting PCR Product and Nucleic AcidHybridization, PCR Meth. Appl. 4: 357-362, dual FRET probes, Espy, M. J.et al. (2002), Detection of Vaccinia Virus, Herpes Simplex Virus,Varicella-Zoster Virus, and Bacillus anthracis by LightCycler PolymeraseChain Reaction after Autoclaving: Implications for Biosafety ofBioterrorism Agents, Mayo Clin. Proc. 77: 624-628, or molecular beaconprobes, Tyagi, S. and Kramer, F. R. (1996), Molecular Beacons: Probesthat Fluoresce upon Hybridization, Nature Biotechnol. 14: 303-308;Tyagi, S. et al. (1998), Multicolor Molecular Beacons for AlleleDiscrimination, Nature Biotechnol. 16: 49-53. Real-time multiplex assaysutilizing PCR amplification have been demonstrated with TaqMandual-labeled linear probes and the 5′ nuclease detection process and,alternatively, with PCR amplification and molecular beacon probes,Tyagi. S. et al. (1998), supra; Vet, J. A. et al. (1999), MultiplexDetection of Four Pathogenic Retroviruses Using Molecular Beacons, Proc.Natl. Acad. Sci. USA 96: 6394-6399; El-Hajj, H. et al. (2001), Detectionof Rifampin Resistance in Mycobacterium tuberculosis in a Single Tubewith Molecular Beacons, J. Clin. Microbiol. 39: 4131-4137.Fluorescence-based multiplex assays currently are limited to about eighttargets per sample by the need to minimize overlaps in emission spectraof fluorophores and, hence, are not expandable for use as highlymultiplexed screening assays.

Highly multiplexed assays rely on spatial segregation of targets forsignal resolution. Spatial segregation enables the use of coding schemesinvolving combinations of differently colored fluorophores(combinatorial coding), combinations of different amounts of eachfluorophore (ratio coding), and both. One example of an assay withspatial segregation is fluorescence in situ hybridization, or FISH, forchromosomal analysis. Speicher et al. (1996), Karyotyping HumanChromosomes by Combinatorial Multi-fluor FISH, Nature Genet. 12:368-375, for example, report the use of 27 probes combinatoriallylabeled using a set of six different fluorophores for analysis ofchromosome spreads. Similarly, segregation of transcription sites incell nuclei has enabled combinatorial coding utilizing multiple, singlylabeled probes per site, as well as ratio coding. Singer, R. H.,International (PCT) patent application WO 00/65094; Levsky, J. M. et al.(2002), Single-Cell Gene Expression Profiling, Science 297: 836-840.Another spatial-segregation probe technique is the use of multiplexprobe arrays, including arrays on DNA chips. Schena, M. et al. (1995),Quantitative Monitoring of Gene Expression Patterns with a ComplementaryDNA Microarray, Science 20: 467-470; Gingeras, T. R. et al. (1998),Simultaneous Genotyping and Species Identification Using HybridizationPattern Recognition Analysis of Generic Mycobacterium DNA Arrays. Genet.Res. 8: 435-448; Han et al. (2001), Quantum-Dot-Tagged Microbeads forMultiplexed Optical Coding of Biomolecules, Nature Biotechnol. 19,631-635. Another segregation approach is the use if electrophoresis toseparate ligated probe pairs of differing lengths. Tong, A. K. et al.(2001), Combinatorial Fluorescence Energy Transfer Tags for MultiplexBiological Assays, Nature Biotechnol. 19: 756-759. In that method, avariation of the oligonucleotide ligation assay (“OLA”) for SNPdetection, differently labeled probes are ligated to capture probes on atarget, and the hybrids are immobilized, washed, released, separatedfrom one another by electrophoresis, and read for each probes'fluorescent code, which is a combination of colors and ratios. Arraymethods and electrophoretic methods remain technically complex,requiring many separate steps, including amplification, hybridization,washing and analysis.

There is not currently available a homogeneous fluorescencehybridization assay that is suitable for use as a highly multiplexedscreening assay, despite the need for such an assay. During suppressivetreatment of the HIV-1 virus with protease inhibitors, for example, anyof about 30 mutations is likely to proliferate over time and to requirechange in treatment. Hirsch, M. S. et al. (1998), Antiretroviral DrugResistance testing in Adults with HIV Infection, JAMA 279: 1984-1991.Lacking a highly multiplexed screening assay, current practice is tosequence the virus in response to a patient's increase in viral load.Sequencing is made difficult by the fact that the arising mutant is notthe only allele present. During initial diagnosis of a patient withparticular symptoms, for example fever, there is available no highlymultiplexed homogeneous assay to screen for an early indication of oneof numerous possible infectious agents that may be the cause of thepatient's symptoms.

An aspect of this invention is highly multiplexed homogeneous assays forscreening samples for the presence of a target nucleic acid sequencefrom among at least ten, and as many as 60 or more, possible targets,utilizing conventional fluorescence detection equipment and techniques,and fluorescently labeled hybridization probes.

Another aspect of this invention is such screening assays that employtarget amplification, optionally with real-time detection, that arecapable of detecting small amounts of pathogens that may be found inotherwise sterile samples such as blood.

Yet another aspect of this invention is kits and oligonucleotide setsfor carrying out particular screening assays according to thisinvention.

SUMMARY

Assays according to this invention are highly multiplexed assayssuitable for screening. By “highly multiplexed,” I mean that an assay iscapable of detecting any of at least six, preferably at least 10, and incertain embodiments 30-60 and even more possible target sequences.

Assays according to this invention are homogeneous nucleic acid in vitroassays fluorescently labeled utilizing hybridization probes. By“homogeneous”, I mean that nucleic acid targets are detected in solutionwithout segregating targets spatially and without washing away unboundhybridization probes. Fluorescence signal is obtained from the reactionmixture itself. Certain preferred embodiments include targetamplification by, for example PCR or NASBA, which enables detection ofsmall amounts of pathogens in samples. When amplification is used, thereaction vessel, for example a microcentrifuge tube, is hermeticallysealed to prevent contamination of the other samples being tested at thesame time, samples not yet tested, equipment and workers.

Detection in assays according to this invention is by fluorescentlylabeled nucleic acid hybridization probes. Fluorescent labels, whenstimulated emit radiation in the visible, ultraviolet or near infraredportions of the electromagnetic spectrum. Each probe is specific to onetarget among the multitude of possible targets being screened. If thegoal and design of an assay are to distinguish among bacterial species,one will design a probe for a species, for example against a conservedregion of M. tuberculosis, or design a probe that hybridizes tonon-identical sequences of multiple strains of the same species. If thegoal, on the other hand, is to distinguish among strains or mutantalleles, one will design each probe against a variable region to providethe necessary distinction and make the probe strain-specific. Preferredprobes for use in this invention are molecular beacon probes, which maybe made mismatch tolerant or mismatch intolerant, depending on the needof a particular assay. As is known in the art, choice of target sequenceand probe design are balanced to minimize both false negatives and falsepositives.

Multiplex capability is achieved by combinatorial coding, with orpreferably without added ratio coding, utilizing a panel ofdistinguishable fluorophores. In a combinatorial code, each code elementis identified by its unique pattern of different labels, in this casecolors. The combinatorial code includes up to four differentfluorophores per code element. The different fluorophores are from apanel of at least four distinguishable fluorophores, more preferably apanel of 4-8 distinguishable fluorophores. The combinatorial code may beduplex coding in which the code elements comprise two colors, optionallyenlarged by additional code elements of one color. The combinatorialcode may by triplex coding in which the code elements comprise threecolors, optionally enlarged by additional code elements of two colors(triplex code combined with a duplex code), and optionally enlarged bycode elements of one color. The combinatorial code may be quadraplexcoding in which the code elements comprise four colors, optionallyenlarged by additional code elements of three colors, two colors or onecolor, or some combination thereof.

Rather than labeling individual probe molecules with multiplex colors togive the probe the pattern assigned to it as a code element, each probeis subdivided into the required number of portions, and each portion islabeled to emit a different color signal. The portions are then mixed tocreate a probe that, when hybridized to its target, will include theunique color signal pattern of the code element.

For the preferred highly quenched probes such as molecular beaconprobes, each portion will emit a single color. For less preferred probeslabeled with two different interacting fluorophores, such as 5′-nucleaseprobes, one option is to utilize a common short-wavelength fluorophore(which I call the “harvester” fluorophore), vary the long-wavelengthfluorophores (which I call the “emitter” fluorophores), and detect thesignal change in the latter. In that case, each portion will emit asignal in a different color. Another option is the reverse, namely, touse a common emitter fluorophore, vary the harvester fluorophores, anddetect the signal change in the latter. In that case, each portion willalso emit a signal in a different color, but for purposes of coding itis the harvester that becomes the detected emitter. However, a thirdoption is to vary both fluorophores to permit detection of a ratiochange in their emissions, so that the signal from one portion might bethe ration a/b, the ratio from a second portion might be the ratio c/d,and the ratio from a third portion might be e/f, where a, b, c, d, e andf are distinguishable fluorophores. In this case the long-wavelengthfluorophores are varied and can be considered to characterize the signalone obtains from any given probe portion. For ease of understanding,descriptions of combinatorial codes in this application and in thefigures generally describe embodiments in which one detects for eachprobe portion the emission of a particular color, which can becharacterized as the emitter fluorophore. It is to be understood thatthe teachings also apply to embodiments utilizing ratios, and personsskilled in the art will be able to apply the teachings to thoseembodiments.

If one begins with a panel of eight different fluorophores, for example,and chooses to subdivide each probe into four portions and to label eachof the four portions with a different color, the codebook can include asmany as 70 different combinations; if one begins with the same-sizedpanel of fluorophores but subdivides each probe into three portions andlabels each of the three portions with a different color, the codebookcan include as many as 56 different combinations; and if one begins withthe same-sized panel of fluorophores but subdivides each probe into twoportions and labels each of the two portions with a different color, thecodebook can include as many as 28 different combinations. Four-color,three-color and two-color codes may be combined with each other and evenwith a one-color code, but my most preferred embodiments do not includesuch combinations. In ratio coding differing amounts of the same labelare used for differentiation. Combinatorial codes according to thisinvention may be augmented with ratio coding, preferably integral ratiocoding, that is, ratios of whole numbers such as 2:1, 3:1, 4:1 or 5:1,and vice versa. It is preferred that portions of molecular beacon probesbe labeled with a non-fluorescent quencher and one signature-colorfluorophore, most preferably without added ratio coding for reasons thatwill be explained.

As stated above, assays according to this invention are homogeneousdetection assays. There is no segregation of targets. There is noseparation of unbound probes from probes bound to their targets.Consequently, in assays according to this invention there must be adetectable signal change indicative of a probe hybridizing to itstarget. Preferred molecular beacon probes are highly quenched whenfree-floating at the detection temperature in the assay but changeconformation and fluoresce when hybridized to their respective targets.Another type of probe whose fluorescence is restored by hybridization toits target and that is generally suitable for use in assays according tothis invention is a “yin-yang” probe, a bimolecular probe labeled with afluorophore on a target complementary strand and a quencher on acompeting strand. Li, Q. et al. (2002), A New Class of HomogeneousNucleic Acid Probes Based on Specific Displacement Hybridization,Nucleic Acids Res. 30: e5. Other probe-assay schemes result in afluorescent signal change indicative of probes hybridizing to targets.In the 5′-nuclease detection process, end-labeled linear (orrandom-coil) probes with two different fluorophores interact byfluorescence resonance energy transfer (FRET) when free-floating but arecleaved during primer extension in a polymerase chain reaction (PCR)amplification, resulting in a signal change (emission from the shorterwavelength fluorophore increases, emission from the longer wavelengthfluorophore decreases, and the ratio of the two changes accordingly).Livak, K. J. et al. (1995), supra. Another system, so-called LightCyclerprobes, works in the opposite manner. Each probe is a pair offluorescently labeled linear oligonucleotides that hybridize adjacentlyon a target and interact by FRET, thereby producing a signal change(emission from the shorter wavelength fluorophore decreases, emissionfrom the longer wavelength fluorophore increases, and the ratio of thetwo changes accordingly). Espy, M. J. et al. (2002), supra. For FRETsystems, I refer to the shorter wavelength fluorophore as a “harvester”and the longer wavelength fluorophore as an “emitter”. However, becausethe background resulting from FRET quenching is considerably higher thancontact-mediated quenching that is commonly used in molecular beaconprobes and yin-yang probes (Tyagi et al. (1998), supra; Marras, S. A. E.et al. (2002), Efficiencies of Fluorescence Resonance Energy Transferand Contact-Mediated Quenching in Oligonucleotide Probes, Nucleic AcidsRes. 30: e122), FRET quenching is not preferred for screening assaysaccording to this invention. Assays according to this inventionpreferably employ probes that are quenched by contact-mediatedquenching, such as molecular beacon probes or yin-yang probes. My mostpreferred probes are molecular beacon probes that include anon-fluorescent quencher. These probes are most preferred, not onlybecause of their low background signal, but also because of their easeof design for a multiplex system. Bonnet, G. et al. (1998),Thermodynamic Basis of the Enhanced Specificity of Structured DNAProbes, Proc. Natl. Acad. Sci. USA 96: 6171-6176.

Assays according to this invention include, but are not limited to,embodiments that employ target amplification. Several exponentialamplification techniques are well known. Among them are the polymerasechain reaction (PCR), which includes thermal cycling, and isothermalamplification schemes such as nucleic acid sequence-based amplification(NASBA), strand-displacement amplification (SDA), transcription-mediatedamplification (TMA), rolling-circle amplification (RCA), andramification amplification methodology (RAM). Amplification assayembodiments may utilize end-point detection or real-time detection. Itis preferred to perform amplification in a sealed environment, forexample sealed tubes, in the presence of the hybridization probes tominimize the possibility of cross contamination between samples thatresults from opening sample tubes.

While not limited by sample source, assays according to this inventionadvantageously may utilize human or animal samples sometimes referred toas being normally sterile, such as blood, tissue or spinal fluid. Suchsamples are advantageous as compared, for example, to sputum, stool orenvironmental samples, because they will contain fewer extraneous DNAsequences that could potentially interfere with amplification anddetection. It is known that at least small amounts of many pathogens canbe found in a patient's blood, even in septic syndromes resulting frombacterial infections. It is preferred to utilize normally sterilesources rather than non-sterile sources where a particular screeningassay permits a choice.

Certain amplification assays according to this invention mayadvantageously take advantage of primers that amplify all or a number ofnucleic acid targets being screened. When screening for mutations, itmay be that numerous possible mutations occur in a particular variablenucleic acid sequence of interest, and one pair of primers flanking thatregion can be used to amplify all such possible mutations. Certain ofthese so-called “universal primers” bind to conserved regions ofmultiple species and are used for amplifying numerous targets, forexample genes of various bacteria. See, for example, Kox, L. F. F. etal. (1995), PCR Assay Based on DNA Coding for 16S rRNA for Detection andIdentification of Mycobacteria in Clinical Samples. J. Clin. Microbiol.33: 3225-3233; Iwen, P. C. et al. (1995), Evaluation of NucleicAcid-Based Test (PACE 2C) for Simultaneous detection of Chlamydiatrachomatis and Neisseria gonorrhoeae in Endocervical Specimens, J.Clin. Microbiol. 33: 2587-2591; Yang, S. et al. (2002), QuantitativeMultiprobe PCR Assay for Simultaneous Detection and Identification toSpecies Level of Bacterial Pathogens, J. Clin. Microbiol. 40: 3449-3454;Schonhuber, W. et al. (2001), Utilization of mRNA Sequences forBacterial Identification, BMC Microbiol. 1: 2; Wong, R. S. and Chow, A.W. (2002), Identification of Enteric Pathogens by Heat Shock Protein 60kDa (HSP 60) Gene Sequences, FEMS Microbiol. Lett. 206: 107-113. Onepair, more likely two or three pairs, of primers may suffice forparticular screening assays.

This invention includes assay kits for performing particular multiplexscreening assays according to this invention. Kits include at least theintended complement of detection probes. We refer to collections ofoligonucleotides as “oligonucleotide sets.” When the assay is anamplification assay, for example a PCR assay, the oligonucleotide setspreferably also include the primers needed for amplification. They mayalso include any amplifiable control sequence. Kits may includeadditional assay reagents such as enzymes, amplification buffer and, ifdesired, sample preparation reagents for isolation of nucleic acids;that is, up to all reagents needed for performing the assay, from samplepreparation through detection.

Fluorescence-based screening assays of this invention are generallyadaptable to detection instruments, which may include thermal cyclingcapability. Instruments may use a single-wavelength source or amulti-wavelength source, for example a white light combined withselectable filters, multiple laser sources or multiple light-emittingdiode sources. Detection instruments vary in their ability todistinguish emissions from multiple fluorophores, which needs to betaken into account when selecting fluorophore panels for a particularassay. In preferred embodiments of assays according to this invention,emissions from fluorophores for each probe are balanced in intensitysuch that intensities from the subdivided portions are closely spaced,which aids identification. Fluorophores vary in intensity, targetsequences vary in accessibility for probe hybridization, and instrumentsmay vary in their responses to different wavelengths. Relative amountsof portions may be varied to achieve balance. Where possible, it ispreferred that all portions for all probes be balanced to further aid inidentification. The results of these assays can be automatically decodedand interpreted by computer programs in real time, enabling the assaysto be highly accurate, and to be carried out in an automated,high-throughput fashion in clinical diagnostic settings.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a table showing the numbers of distinguishable combinationsobtained using two or three colors per probe and panels of 4-8distinguishable fluorophores.

FIG. 2 illustrates fluorescence emissions of a real-time amplificationassay in which a single target sequence is present in the sample beingtested (out of many possible target sequences that the assay is designedto detect) and one probe (labeled with three different colors of equalfluorescence intensity) among the many different probes in the assaymixture binds to the amplified target sequence.

FIG. 3 illustrates fluorescence emissions of a real-time amplificationassay in which two different target sequences are present at differentconcentrations in the sample being tested and two probes (each labeledwith a different combination of three different colors of equalfluorescence intensity, no color being shared by both probes) bind tothe amplified target sequences.

FIG. 4 illustrates fluorescence emissions of a real-time amplificationassay in which two different target sequences are present at differentconcentrations in the sample being tested and two probes (each labeledwith a different combination of three different colors of equalfluorescence intensity, one color being shared by both probes) bind tothe amplified target sequences.

FIG. 5 illustrates fluorescence emissions of a real-time amplificationassay in which two different target sequences are present at the sameconcentration in a sample being tested, and two probes (labeledaccording to the first ternary color codebook shown in FIG. 6 a) sharetwo colors, leading to unambiguous interpretation of the results.

FIGS. 6 a and 6 b contain tables that show the effect of alternativeprobe coding schemes for detecting ten different possible targets in asample, utilizing probes that are each labeled with three differentcolors of equal fluorescence intensity from a palette of five differentfluorophores, on the results that would be obtained for every one of the45 possible combinations of two different targets that could be presentat the same concentration in a sample being tested.

FIG. 7 illustrates fluorescence emissions of a real-time amplificationassay in which two different target sequences are present at the sameconcentration in a sample being tested, and two probes (labeledaccording to the first ternary color codebook shown in FIG. 6 a) shareone color, leading to an ambiguous result.

FIG. 8 illustrates fluorescence emissions of a real-time amplificationassay in which the same sample used in the assay shown in FIG. 7 isretested using probes labeled according to the second ternary colorcodebook shown in FIG. 6 b, providing an example of how a second assayutilizing an alternative coding scheme can resolve ambiguous results.

DETAILED DESCRIPTION

FIG. 1 is a table presenting certain combinatorial coding schemes usefulin this invention. The top portion of the table presents the number ofcodes obtained by having each probe differently labeled with twodifferent fluorophores from panels of 4, 5, 6, 7 or 8 fluorophores. Ifthe panel includes eight fluorophores, there are 28 possible codes. Thebottom portion of the table presents the number of codes obtained byhaving each probe labeled with three different fluorophores from thesame panels of 5, 6, 7 or 8 fluorophores. If the panel includes eightfluorophores, there are 56 possibilities. Assays according to thisinvention utilize two or three (and sometimes four) differently labeledportions of each probe and a panel of fluorophores of sufficient size topermit at least six differently coded probes. This may be accomplished,for example, by combining the two portions of the table, that is, usingboth codes of two colors and codes of three colors. For example, probesmay be subdivided into three portions for use with a panel of fivefluorophores, providing up to ten differently coded probes, as outlinedin the bottom portion of the table, and additional probes may besubdivided into two portions for use with a panel of five fluorophores,providing up to ten additional differently coded probes, as outlined inthe top portion of the table. In this manner, as many as 20 differentlycoded probes can be combined in the same assay mixture.

Assays according to this invention utilize one probe (which may be asingle-molecule probe, such as a molecular beacon probe or a TaqManprobe, or a bi-molecular probe, such as a yin-yang probe or aLightCycler probe pair) for each target. Coding is achieved, except fora code element represented by a single color, by subdividing probes intoportions and labeling the portions according to a combinatorial code orcombinatorial-plus-ratio code from a panel of at least four, preferably5-8, distinguishable fluorophores. Utilizing our preferred molecularbeacon probes for purposes of illustration, each probe portion isdesigned to have a different color signature. Certain of the probes maybe wavelength-shifting probes, that is, probes in which one arm islabeled with a quencher and the other arm is labeled with a FRET paircomprising a “harvester” fluorophore to absorb energy from thestimulating light source and transfer that energy to an “emitter”fluorophore that fluoresces strongly at its own characteristic longerwavelength spectrum when the probe is in its open configuration. Weutilize wavelength-shifting probes with instruments that employ a singlewavelength source that is not highly absorbed by fluorophores that emitvisible fluorescence at longer wavelengths, for example, Texas Red. SeeTyagi, S. et al. (2000), Wavelength-Shifting Molecular Beacons, NatureBiotechnol. 18: 1191-1196; El-Hajj, H. et al. (2001), supra. It is alsopossible to utilize one fluorophore to quench another fluorophore bycontact-mediated quenching. This creates two additional possibilities.First, a fluorophore that has relatively low emission in the systembeing used could be placed on each arm so that the fluorophores quenchone another when the probe is closed but both fluoresce with the samecolor, increasing its intensity, when the probe is hybridized to itstarget and open. This technique can be used to add ratio coding to acombinatorial code. Second, one portion of a probe can carry two codeelements (colors) by placing different fluorophores on the two arms, sothat the fluorophores quench one another when the probe is closed buteach fluoresces in its own characteristic emission spectrum when theprobe is hybridized to its target and open. This can be used to combinetwo portions into one and thereby reduce the number of differentportions needed for a two-color, three-color or four-color code element.It is equivalent to having separate portions. Because quenching bynon-fluorescent quenchers is more efficient than quenching byfluorophores and because having two emitting fluorophores on the samemolecule removes the option to balance their intensities by means ofrelative concentration, preferred embodiments of this invention utilizea non-fluorescent quencher on one arm of molecular beacon probes. Dabcylis a preferred quencher, as is Black Hole Quencher No. 2.

“Codebooks” useful in this invention utilize four colors per probe,three colors per probe, two colors per probe, or combinations of four,three or two colors per probe, with or without single-color probes.Preferred codebooks utilize either only four colors per probe, onlythree colors per probe or only two colors per probe. The reason theseare preferred is that the appearance of, for example, one color arisingat a particular PCR cycle signals that an error has been made in assaypreparation. If single-color probes are used, such an error is notobvious. In some embodiments of this invention, combinatorial coding maybe supplemented with ratio coding to increase the number of codeelements or possibilities. Preferred ratios are integral, from 5:1 to1:5, preferably from 3:1 to 1:3. Ratio coding is most appropriate forscreening assays in which it is highly likely that only one target willbe found. The reason for this is that when the code is strictlycombinatorial and two or more targets are present, changes in intensitylevels are helpful for resolving ambiguities that may arise in analysisof the emission spectrum. Therefore, preferred assays that are nothighly likely to find only one target utilize strictly combinatorialcoding.

Various fluorophores can be included in panels of fluorophores. Certainfluorophores have relatively narrow emission spectra, for example, Vic,Alexa 488, and quantum dots (Gao et al. (2002), Quantum-Dot Nanocrystalsfor Ultrasensitive Biological Labeling and Multicolor Optical Encoding,J. Biomed. Opt. 7, 532-537), making them particularly useful for highlymultiplexed assays utilizing panels of more than six fluorophores.

Oligonucleotide probes, including molecular beacon probes, areconveniently prepared by solid-phase DNA synthesis on, for example, anApplied Biosystems 394 DNA/RNA synthesizer. DNA nucleotides or RNAnucleotides may be used, including modified nucleotides such as2′-O-methylribonucleotides, which yield strands resistant to cleavage.Our preferred method of molecular beacon synthesis starts withcontrolled-pore glass columns containing the quencher dabcyl forincorporation at the 3′ end of the probe. These columns are availablefrom Biosearch Technologies (Novato, Calif., USA). Our preferred methodof incorporating fluorophores at the 5′ end of the probe is to utilizethiolmodifier phosphoramidites or aminomodifier phosphoramidites so thatiodoacetylated fluorophore derivatives can be coupled to the 5′-thiolgroups and succinimidyl esters of fluorophores can be coupled to the5′-amino groups. For tetrachlorofluorescein, our practice is toincorporate directly a tetrachlorofluorescein phosphoramidite. Forwavelength-shifting probes a non-terminal fluorophore is required. Wehave used fluorescein phosphoramidites to incorporate internalfluorescein moieties for this purpose. Our molecular beacon probesynthesis includes probe purification by gel exclusion chromatographythrough NAP-5 Sephadex columns, followed by HPLC through a reverse-phasecolumn such as the C-18 column marketed by Waters. Finally, probes areprecipitated with ethanol and dissolved in 100 μl Tris-EDTA buffer.Molecular beacon probes should have a signal-to-noise ratio of at least25, preferably closer to 100, keeping in mind that for a triplet code,for example, each fluorophore will be common to 30-60% of the probes.

As indicated above, the probes are subdivided into two, three or fourportions for labeling and use. It is advantageous that the variousportions of a probe, and where possible all portions in the assay,produce roughly the same signal intensity. Accordingly, our preferredassays and kits have portions “balanced” for that purpose. By “balanced”I mean that the fluorescence intensities of the signals from thedifferent portions are generally within 20% of one another, mostpreferably within 5%. The fluorescence intensity produced by aparticular probe portion, for example a molecular beacon probe portion,will depend on the intrinsic properties of its emitter fluorophore (and,if it is a wavelength-shifting molecular beacon probe, also itsharvester fluorophore), the nature of the target, as some targets acceptprobes more readily than others, and quite possibly on the instrumentutilized for detection. By testing a probe against its target in theassay on the instrument to be used, the amounts of the portions relativeto one another can be adjusted to produce balanced signals. I preferfurther that all portions of all of the combinatorially labeled probesbe balanced. After the portions of each probe are balanced, the amountsof the probes relative to one another can be adjusted to producebalanced signals. It is possible to adjust signal readings by adjustingthe detection instrument rather than the amounts of the portions of eachprobe to account for differences in the fluorophores and the instrument.For example, if a probe with three portions labeled a, b, c producesrelative intensities with equal volumes of each portion of 1.0, 1.5 and3.0, respectively, the instrument could be programmed to multiplyreadings from portion “a” by three, from portion “b” by two, and fromportion “c” by one. Then only the relative amounts of the probes wouldhave to be balanced. My preference, however, is to adjust the portionamounts rather than the instrument. Utilizing balanced emissions makesinterpretation by a human observer significantly easier, as one can seeimmediately whether or not three colors, for example, indicate aparticular code element.

Assays according to this invention are highly multiplexed. While notlimited as to sample source, the assays are particularly applicable foruse with samples from human or other animal body sites that areconsidered sterile, such as blood, tissue and cerebral spinal fluid.Non-sterile specimens, such as sputum or stool, are likely to havenucleic acids from a variety of sources and are more difficult to screenfor that reason. Pathogens generally infect the blood stream to at leasta small degree. However, nucleic acid amplification assays requireminimal amounts of target sequence in the sample. For example, Kane etal. compared blood culturing to PCR assays utilizing “universal’bacterial primers to diagnose bacterial infection in critically illsurgical patients. Kane, T. D. et al. (1998), The Detection of MicrobialDNA in Blood: a Sensitive Method for Diagnosing Bacteremia and/orBacterial Translocation in Surgical Patients. Ann. Surg. 227: 1-9.Whereas blood cultures were positive for only 14% of the samples, thePCR assays were positive for 64%.

For embodiments employing amplification, such as PCR or NASBA, primersare required for the various possible targets. Primer design is known inthe art. Primers can be tested with other primers and then with probesto check for undesired interactions. It is important that primers, andto a lesser extent probes, not interact in multiplex reactions. A typeof high-specificity primer useful in this invention is disclosed inpublished international patent application WO 00/71562. Such primersform hairpin stems that significantly reduce undesired hybridizations.We have decreased primer interactions by utilizing such high-specificityprimers, taking care to ensure that the melting temperature (T_(m)) ofthe stem is approximately 6° C. above the primer annealing temperatureutilized in the assay. We have constructed two pairs of universalprimers that together amplify a segment of the 16s ribosomal RNA gene ofmany bacterial species. One pair includes 5′-TGACGACAACCATGCACC-3′ (SEQ.ID NO. 1) and 5′-ATGTGGTTTAATTCGAAGCAA-3′ (SEQ. ID NO. 2) to amplifytarget regions in B. anthracis and related gram-positive bacterial. Theother pair includes 5′-GTGGACTTAGATACCCTGGTAGTCCAC-3′ (SEQ. ID NO. 3)and

5′-GCGTTGCATCGAATTAA-3′ (SEQ. ID NO. 4), which amplify target regions inseveral important gram-positive bacteria and gram-negative bacteria. Theunderlined sequences of the third identified probe are hairpin-formingsequences added to reduce undesired hybridizations by this primer.

Amplification assays with real-time detection can be performed on avariety of instruments that utilize different techniques for excitingfluorophores and detecting emissions. Excitation can be by lasers, whitelight coupled with filters, or light-emitting diodes. In addition, theassay instrument may utilize a synchronous scanning mode, in whichnarrow excitation and emission wavelengths are continuously varied,while maintaining a fixed wavelength difference between them. Lee et al.(1999), Seven-Color, Homogeneous Detection of Six PCR Products,Biotechniques 27, 342-349. The choice of instrumentation may affect thenumber of different fluorophores that can be included in a panel.

Screening assays according to this invention, including but not limitedto real-time amplification assays, may be positive for a single target,in which case the target is identifiable by its unique signature,preferably its unique color signature but possibly by itscolor-plus-intensity signature. On occasion two or even more than twotargets may be present in a sample. Deciphering the spectrum in such acase may take advantage of the fact that, although the absolutefluorescence of a probe is dependent on target concentration, thefluorescence ratios of two probe portions hybridizing to the same targetwill be independent of target concentration. Taking the case of strictlycombinatorial coding utilizing three-color codes wherein the intensitiesof the portions have been balanced, assume that two targets are present.If the targets share no color, there will result six colors. However,because equal concentrations of the two targets in the sample are highlyunlikely, direct probing of the sample is likely to result in threecolors of one intensity and three colors of a second intensity, therebyidentifying the two targets. If the assay, on the other hand, is areal-time amplification assay, the times at which signals rise abovebackground, for example the threshold cycle (C_(T)) of a PCR reaction,are likely to differ, leading to two groups of three colors, each grouparising at a different time, thereby identifying the two targets.

If the codes assigned to the probes for the two targets share a color,in an assay based on directly probing the nucleic acids in a sample, thefluorescence intensity for that color will not match the fluorescenceintensity for the other colors. Rather it will be a combination from twotargets and will stand out from the level of the other colors, therebyidentifying it as the color in common. If the assay, on the other hand,is a real-time amplification assay, three colors will come up togetheras signal from the more abundant target. When the second threshold forthe less abundant target is reached, two more colors will come uptogether and the intensity curve for the common color will change slopeto a higher value, thereby identifying it as the color in common.

Taking the case where ratio coding is also used, assume one color iscommon to two targets, but its intensity ratio for the more abundanttarget is 2:1 as compared to the other portions for that target. In areal-time amplification assay three colors will rise above background asthe first threshold is reached, but one color (the common color) willhave a higher slope than the other two colors. Nonetheless, when thesecond threshold is reached, the intensity curve of the common colorwill change slope to a higher value, signifying its commonality.

When two different target species are present in the sample at the sameconcentration, the colors encoding each probe will appear at the sametime. In some instances of this situation it may not be possible tounambiguously identify each code. In such an instance a repeat assayutilizing an alternative coding scheme for the targets, should in almostall cases resolve the ambiguity.

EXAMPLE 1

In this example, a real-time PCR amplification assay is carried out onDNA isolated from a normally sterile blood sample suspected ofcontaining an infectious bacterium. The sets of primers that are presentin the reaction mixture enable the amplification of a segment of the 16sribosomal RNA gene that is present in all of the bacterial species thatare suspected of possibly being present in the blood sample. Amultiplicity of molecular beacon probes is present in the assay, one foreach of the suspected bacterial target species. Each different molecularbeacon possesses a universal quencher moiety (dabcyl) covalently linkedto its 3′ end. Each different species-specific molecular beacon probe islabeled combinatorially according to this invention with a unique set ofthree differently colored fluorophores. These fluorophores arecovalently linked to the 5′ end of each oligonucleotide. For everyspecies-specific probe that is present in the assay mixture, theabundance of each of the three differently colored oligonucleotides thattogether make up the species-specific probe is adjusted prior toperforming the assay so that the fluorescence intensity of each of thethree colors of that probe will be approximately the same when measuredin the assay instrument used to carry out the assay. FIG. 2 is a plot ofresults that will be obtained in this assay. The “x” axis represents thenumber of amplification cycles performed by the time that eachmeasurement of fluorescence is made. This measurement is madeautomatically by the assay instrument in a manner that permits thefluorescence intensity of each of the differently colored fluorophoresthat are used to label the probes to be measured. The “y” axisrepresents the fluorescence intensity due a particular coloredfluorophore. Measurements are taken during the annealing phase of eachamplification cycle. Measurements are taken in real time during eachamplification cycle throughout the course of the amplification. Thefluorescence intensity of each of the differently colored fluorophoresused to label the probes is plotted as a function of the number ofamplification cycles completed.

In the experiment shown in FIG. 2, a signal appears that is sufficientlyintense to be detected above the background fluorescence after aparticular number of cycles of amplification have been carried out. Theamplification cycle at which this occurs is known as the “thresholdcycle”, generally abbreviated as “C_(T)”. The signal that appears afterthe threshold cycle consists of three different colors (a, b, and c).This signal rises in a linear fashion as more cycles of amplificationare completed. The set of three colors that appear together incombination identify which bacterial species is present in the bloodsample. The abundance of the species in the sample is determined fromthe threshold cycle at which the signal arises. The threshold cycle isinversely proportional to the logarithm of the number of targetmolecules in the sample

EXAMPLE 2

In this example, an assay is carried out in the same manner and for thesame purpose as the assay described in Example 1. The results that willbe obtained in this experiment are shown in FIG. 3. Two differentsignals occur, one (consisting of colors a, b, and c) appears early inthe reaction, and the other (consisting of colors d, e, and f) appearslater in the reaction. This result indicates that two differentbacterial species are present in the sample, one (identified by colorsa, b, and c) is relatively abundant, and the other (identified by colorsd, e, and f) is relatively rare and therefore requires more cycles ofamplification before there is sufficient amplified product for thefluorescence signal due to the hybridization of the species-specificprobe to be seen above the background fluorescence. The unique set ofcolors in each of the two signals unambiguously identifies each of thetwo bacterial species that are present in the sample.

EXAMPLE 3

In this example, an assay is carried out in the same manner and for thesame purpose as the assay described in Example 1. The results that willbe obtained in this experiment are shown in FIG. 4. Two differentsignals occur, one (consisting of colors a, b, and c) appears early inthe reaction, and the other (consisting of colors d and e) appears laterin the reaction. In addition, the slope of the curve showing thefluorescence intensity of color “c” increases after the threshold cycleat which the later signal (consisting of colors d and e) occurs. Thisresult indicates that two different bacterial species are present in thesample, one (identified by colors a, b, and c) is relatively abundant,and the other (identified by colors c, d, and e) is relatively rare andappears later. The increase in the slope of the curve showing thefluorescence intensity of color “c” is due the contributions of “c”fluorescence from probes binding to amplified segments of the DNA fromthe rare bacterial species being added the “c” fluorescence from probesbinding to amplified segments of the DNA from the abundant bacterialspecies. Even though the three-color codes for each of the two bacterialspecies that are present in the sample have one color in common, theoccurrence of the two species at different concentrations in the sampleenables the signals from each species to be distinguished from oneanother. In general, when two (or even three) species are simultaneouslypresent in a clinical sample, each will occur at a differentconcentration, and the coded signal due to the presence of each of thebacterial species can be distinguished from the signals due the otherspecies in the sample.

EXAMPLE 4

This example illustrates how unambiguous results can be obtained eventhough two different species are present in a sample at approximatelythe same concentration. The assay described in this example is carriedout in the same manner and for the same purpose as the assay describedin Example 1. In this experiment, the probes present in the assaymixture are designed to detect ten different bacterial species(designated species 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9). Three-color codesare used to label each of the ten probes, from a palette of fivedifferently colored fluorophores (designated a, b, c, d, and e). Forevery species-specific probe that is present in the assay mixture, theabundance of each of the three differently colored oligonucleotides thattogether make up the species-specific probe is adjusted prior toperforming the assay so that the fluorescence intensity of each of thethree colors of that probe will be approximately the same when measuredin the assay instrument used to carry out the assay. For this assay, thefollowing coding scheme (which I call the “first ternary colorcodebook”) is used:

probe for species 0 abc probe for species 1 abd probe for species 2 abeprobe for species 3 acd probe for species 4 ace probe for species 5 adeprobe for species 6 bcd probe for species 7 bce probe for species 8 bdeprobe for species 9 cde

The results that will be obtained in the experiment carried out withprobes labeled according to this codebook are shown in FIG. 5. A signalconsisting of four different colors (a, b, c, and d) arises at the samethreshold cycle. The slope of the curves for two of the colors (a and b)is twice as high as the slope of the curves for the other two colors (cand d). This result indicates that two different bacterial species arepresent in the sample at approximately the same concentration, and thatthe three-color codes of their respective probes have two colors incommon. Since the slope of the curves for colors “a” and “b” isapproximately twice as high as the slope of the curves for colors “c”and “e”, colors “a” and “b” are components of the codes for bothbacterial species that are present in the sample. The conclusion to bedrawn from these results is that one of the probes that bound toamplified target sequences was encoded “abc” and the other probe thatbound to amplified target sequences was encoded “abd”. By comparingthese results with the coding scheme in the first ternary color codebook(shown above), “abc” is seen to be the code for bacterial species “0”and “abd” is seen to be the code for bacterial species “1”. Thus, theresults unambiguously indicate that the sample contains species “0” andspecies “1” in approximately equal concentration.

EXAMPLE 5

This example illustrates how ambiguous results can sometimes be obtainedwhen two different species are present in a sample at approximately thesame concentration. The assay described in this example is carried outin the same manner and for the same purpose as the assay described inExample 4. The probes used in this assay utilize the first ternary colorcodebook (shown in Example 4).

FIG. 6 a contains a two-part table (based on the first ternary colorcodebook) that shows the results that should occur when two differentspecies are present in a sample at the same concentration for each ofthe 45 combinations of two species that are possible. In 30 of thesecombinations four colors arise, two at a higher rate of fluorescenceintensity increase (identified by underlined letters) and two at a lowerrate of fluorescence intensity increase (identified by letters that arenot underlined). Each of these 30 combinations is unique within thetable, therefore providing an unambiguous result.

However, from an examination of FIG. 6 a it can be seen that in 15 ofthe 45 possible combinations of two different species that can bepresent in a sample at the same concentration five colors arise, one ata higher rate of fluorescence intensity increase (identified by anunderlined letter) and four at a lower rate of fluorescence intensityincrease (identified by letters that are not underlined). Thesecombinations, however, are not unique. For example, the code “abcde”occurs three times in the upper table: once for the combination ofspecies “2” and “9”, a second time for the combination of species “4”and “8”, and a third time for the combination of species “5” and “7”. Anexample of an experiment that yields such an ambiguous outcome is shownin FIG. 7. The results of this assay show five colors arising at thesame threshold cycle (a, b, c, d, and e), one color of which (e)increases in intensity at twice the rate as the other four colors.

EXAMPLE 7

The ambiguities inherent in results such as those shown in FIG. 7 can beresolved by repeating the assay with the same probes encoded by analternative coding scheme. For example, the assay giving results shownin FIG. 8 is carried out on the same sample used to obtain the resultsshown in FIG. 7, except that the probes are encoded by the followingalternative coding scheme (which I call the “second ternary colorcodebook”):

probe for species 0 cde probe for species 1 bde probe for species 2 bceprobe for species 3 bcd probe for species 4 ade probe for species 5 aceprobe for species 6 acd probe for species 7 abe probe for species 8 abdprobe for species 9 abc

FIG. 8 shows the results that will be obtained from an assay on the samesample used in the experiment shown in FIG. 7, except that the probesare encoded according to the second ternary color codebook. A signalconsisting of four different colors (a, b, c, and e) arises at the samethreshold cycle. The slope of the curves for two of the colors (b and c)is twice as high as the slope of the curves for the other two colors (aand e). Since the slope of the curves for colors “b” and “c” areapproximately twice as high as the slope of the curves for colors “a”and “e”, colors “b” and “c” are present in the codes for both bacterialspecies that are present in the sample. The conclusion drawn from theseresults is that one of the probes that bound to amplified targetsequences is encoded “abc” and the other probe that bound to amplifiedtarget sequences is encoded “bce”. By comparing these results with thecoding scheme in the second ternary color codebook (shown above), “bce”is seen to be the code for bacterial species “2” and “abc” is seen to bethe code for bacterial species “9”. Thus, the results shown in FIG. 8resolve the ambiguity posed by the results shown in FIG. 7,unambiguously indicating that the sample contains species “2” andspecies “9” in approximately equal concentration.

FIG. 6 b shows the results that should occur for all 45 differentcombinations of two different bacterial species in a sample atapproximately the same concentration when the probes are encodedaccording to the second ternary color codebook. A comparison of thetwo-part table in FIG. 6 a with the two-part table in FIG. 6 b showsthat all 45 possible combinations of two different bacterial species canbe unambiguously identified by carrying out a second assay withalternatively coded probes when it is necessary to resolve ambiguitiesarising from the first assay.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A highly multiplexed homogeneous in vitro assay to screen a samplefor the presence of any of a minimum of six different nucleic acidtargets comprising a) contacting the sample with a set of at least sixfluorescent hybridization probes, each one specific for a different oneof said nucleic acid targets, wherein i) each probe in said set issubdivided into the same number of portions, said number being in therange of from two to four, ii) each portion of each subdivided probe islabeled with a different emitter fluorophore from a panel of at leastfour spectrally differentiable fluorophores, iii) each probe in said sethas a unique color signal pattern, and iv) hybridization of each probeleads to a detectable fluorescence signal change indicative of the colorpattern of the labeling of its portions; b) stimulating the fluorophorespresent in the sample; and c) detecting changes in emissions signalsfrom said fluorophores without spatially segregating the nucleic acidtargets from one another to detect the color pattern of one of saidprobes so as to identify which target is present.
 2. The assay accordingto claim 1 further comprising exponentially amplifying said nucleic acidtargets.
 3. The assay according to claim 2 wherein changes in emissionsignals are detected in real time during amplification.
 4. (canceled) 5.The assay according to claim 1 wherein the relative amounts of theportions of each probe are adjusted relative to one another to providebalanced signal changes.
 6. The assay according to claim 1 wherein saidset of probes additionally includes at least one probe that is ratiocoded.
 7. The assay according to claim 1 wherein said probes arequenched probes whose fluorescence is restored by hybridization to theirtargets.
 8. The assay according to claim 7 wherein said probes aremolecular beacon probes having a non-fluorescent quencher on one arm andan emitter fluorophore on the other arm.
 9. The assay according to claim8 wherein the fluorescence intensities of the portions of each probe arebalanced relative to one another.
 10. The assay according to claim 8further comprising exponentially amplifying said nucleic acid targets inthe presence of said probes and detecting increases in fluorescence fromemitter fluorophores in real time.
 11. The assay according to claim 10wherein said panel of fluorophores consists of from four to eightdifferentiable fluorophores, wherein the relative amounts of theportions of each probe are adjusted relative to one another to providebalanced fluorescence emissions.
 12. The assay according to claim 11wherein the relative amounts of all portions of all probes are adjustedrelative to one another to provide balanced fluorescence emissions. 13.A kit of reagents for use in a highly multiplexed in vitro assay toscreen a sample for the presence of any of a minimum of six differentnucleic acid targets comprising a set of fluorescent hybridizationprobes including at least six combinatorially coded fluorescenthybridization probes, each specific for a different nucleic acid target,whose hybridization to their targets can be detected in said assay by afluorescent signal change indicative of their labeling, wherein theprobes in the set are combinatorially coded with up to four differentemitter fluorophores from a panel of at least four spectrallydifferentiable fluorophores by labeling portions of each probe withdifferent emitter fluorophores.
 14. The kit according to claim 13further comprising primers for exponential amplification of said nucleicacid targets.
 15. The kit according to claim 14 further comprisingamplification reagents for amplifying said nucleic acid targetsutilizing said primers.
 16. The kit according to claim 13 furthercomprising at least one probe that is ratio coded.
 17. The kit accordingto claim 13 wherein the combinatorially coded probes are quenched probeswhose fluorescence is restored by hybridization to their targets. 18.The kit according to claim 17 wherein said probes are molecular beaconprobes having a non-fluorescent quencher on one arm and an emitterfluorophore on the other arm.
 19. The kit according to claim 18 whereinthe relative amounts of the portions of each probe are adjusted relativeto one another to provide balanced fluorescence emissions.
 20. The kitaccording to claim 19 wherein the relative amounts of all portions ofall probes are adjusted relative to one another to provide balancedfluorescence emissions.
 21. The kit according to claim 18 furthercomprising primers for exponential amplification of said nucleic acidtargets.
 22. The kit according to claim 21 further comprisingamplification reagents for amplifying said nucleic acid targetsutilizing said primers.
 23. The assay according to claim 1 furthercomprising at least one additional probe that is subdivided into fewerportions than are the probes in said set of probes, wherein said atleast one additional probe has a unique color pattern.